This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this project is to understand the molecular signaling mechanisms that control tumor cell migration, invasion, and metastasis to distinct sites in the body. Cell metastasis is the major cause of disease relapse and decreased patient survival. We have developed a unique biochemical method to purify he very leading front (lamellipodia) of migrating cells(2). This breakthrough technology allows us to identify the key regulatory proteins that facilitate lamellipodia formation which is responsible for mediating cell invasion and metastasis. We will use monkey kidney epithelial cells (COS-7) and metastatic human breast adenocarcinoma cells (commercially available) for these studies. Initial analysis of purified lamellipodia from these cells has revealed that phosphotyrosine (PY) proteins are highly activated in the leading lamellipodia. Pharmacological inhibition of tyrosine phosphorylation inhibits lamellipodia formation indicating that complex signaling cascades operate to control this process through modulation of tyrosine networks. Therefore, our major objective is to characterize the PY proteins (lamellipodia phosphoproteome) responsible for lamellipodia formation and cancer cell metastasis using immunoaffinity purification with anti-phosphotyrosine antibodies followed by proteome analysis to identify proteins of interest. Results from our study will provide valuable information on the signals that control cell migration and metastasis, and provide targets for therapeutic intervention of cancer progression. Our specific aims are: Specific Aim 1. To identify PY proteins and their specific sites of tyrosine phosphorylation in the leading front of migrating cells. Specific Aim 2. To functionally test identified PY proteins using siRNA protein knockdown and site directed mutagenesis of key phosphotyrosine sites identified by MS followed by cell-based assays and animal models of cell migration established in our laboratory. Specific Aim 3. To determine the temporal and spatial distribution of tyrosine-containing proteins, phosphorylated and non-phosphorylated, and their relative abundance in a time course investigation of dormant vs actively migrating cells applying a combination of pulsed stable isotopic labeling with subcellular fractionation of cell bodies and of enriched lamellipodia. Specific Aim 4. To map the putative signaling cascades and develop functional relationships among the PY proteins using bioinformatics and computer modeling systems.